CULTURING CIRCULAR ssDNA VIRUSES FOR THE PRODUCTION OF VACCINES

ABSTRACT

The present invention relates to the use of interferon in the in vitro cultivation of animal circular ssDNA virus such as Porcine Circovirus 2 or human TT virus in an animal cell line. Increased titres of animal circular ssDNA virus are obtained by addition of interferons or agents which ensure the production of endogenous interferons by said cell line and/or by the reduction of endosomal-lysosomal system acidification.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/680,420, filed Feb. 28, 2007, which is a continuation-in-part of U.S.patent application Ser. No. 11/275,842, filed Jan. 31, 2006, which, inturn, claims benefit of U.S. Provisional Application Ser. No.60/649,738, filed Feb. 3, 2005, the disclosures of which areincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to cell culture methods for the in vitrocultivation of viruses (more in particular animal circular ssDNAviruses), which are of use in the production of vaccines, as well as thevaccines produced. The invention further relates to in vitro methods forthe diagnosis of animal circular ssDNA viral infection.

BACKGROUND

Animal circular ssDNA viruses are a group of viruses of pathogenicimportance. Human circular ssDNA viruses have been detected in patientswith hepatitis of unknown aetiology. The titre of human TTV virus isalso significantly higher in HIV-infected patients with AIDS, AIDSpatients with a low CD4 T cell count, or patients with high HIV viralloads [Shibayama et al. (2001) AIDS 15, 563-570]. Touinssi et al. [J.Clin Virol. (2001) 21, 135-141] report a relationship between theprevalence of elevated viral loads of TTV virus and the level ofimmunocompetence of the populations studied and suggest that stimulationof the immune system by an interferon treatment was able to clear TTVviraemia. Moreno et al. (World J Gastroenterol (2004) 1, 143-146)however found that administration of PEG-IFN plus ribovarin could notinduce a TTV sustained response in patients infected with hepatitis C.

Porcine circoviruses are associated with the occurrence of postweaningmultisystemic wasting syndrome (PMWS) in pigs. Porcine circovirus 2(PCV2) is a member of the family of Circoviridae. It is a very smallvirus with a relatively simple structure. The PCV2 viral genome does notcode for a viral DNA-polymerase, making it dependent on cellular enzymesto complete its infectious cycle.

When PCV2 is inoculated in susceptible pigs, a high variation in virusreplication is observed. It has been observed that PCV2 is able toreplicate better in a host that is simultaneously inoculated with otherviruses such as porcine reproductive and respiratory syndrome virus(PRRSV) [Allan et al. (2000) Arch Virol. 145, 2421-2429] or porcineparvovirus (PPV) [Allan et al. (2000) J Vet Med B. 47, 81-94]. A generalstimulation of the immune system with keyhole limpet hemocyanin[Krakowka et al. (2001) Vet Pathol. 38, 31-42] has also been found toameliorate the replication of PCV2 in the host.

It has been shown for PCV2 that inhibition of endosomal-lysosomal systemacidification in a monocytic cell line reduced PCV2 infection (Misinzoet al., 2005. J Gen Virol. 86:2057-68). These data demonstrated thatPCV2 requires an acidic environment for infection of a monocytic cellline.

Many cytokines are capable of modulating the susceptibility of the hostto a viral infection. The most studied and best understood is theanti-viral effect of type I interferons (IFN-alpha and IFN-beta). Butalso other cytokines such as tumour necrosis factor alpha (TNF-alpha)and type II interferon (IFN-gamma) have been shown to influenceinfection. The antiviral effect of interferons has been demonstrated formany viruses and has been found to be so consistent and potent thathumans and animals are routinely administered recombinant interferon(IFN-alpha) for the treatment of viral infections.

SUMMARY OF THE INVENTION

The present invention is based on the surprising observation, that typeI and type II interferons have an enhancing effect on the viral titreobtained in cell cultures after infection in vitro with animal circularssDNA virus, more particularly porcine circovirus 2. In addition it wasobserved that, when cultivating PCV2 in PK-15 epithelial cells in thepresence of inhibitors of endosomal-lysosomal system acidification, theviral titre obtained in the cell cultures is further increased and thisindependently of the effect of interferons. The combination ofinterferons and endosomal-lysosomal system acidification inhibitorsgenerates a synergistic effect.

A first aspect of the present invention relates to the use of aninterferon-containing medium for the cultivation of animal circularssDNA virus in an animal cell line.

According to a first embodiment of this aspect of the invention methodsare provided for the in vitro cultivation of an animal circular ssDNAvirus comprising the step of inoculating cells of a continuous animalcell line in a culture medium with the circular ssDNA virus, therebyensuring that the culture medium contains interferon. Different methodsfor ensuring that the medium contains interferon are envisaged.According to a specific embodiment a method is provided for the in vitrocultivation of an animal circular ssDNA virus comprising the steps of a)inoculating cells of a cell line, more particularly a continuous animalcell line, in culture medium with a circular ssDNA virus and b)administering an exogenous interferon or an agent which induces theendogenous production of an interferon by said cells. In view of thefact that the object of the methods according to this aspect of theinvention is the generation of PCV2 viral particles, the methods of theinvention typically additionally comprise the step of isolating PCV2particles from the medium and/or infected cells of the continuous animalcell line.

A second aspect of the present invention relates to the cultivation ofanimal circular ssDNA virus in an animal cell line, whereby it isensured that endosomal-lysosomal system acidification in the animal cellline is reduced.

Accordingly, the present invention provides methods for the in vitrocultivation of an animal circular ssDNA virus comprising the step of (a)inoculating cells of a continuous animal cell line in a culture mediumwith the circular ssDNA virus, and (b) cultivating the continuous animalcell line thereby ensuring that endosomal-lysosomal system acidificationis reduced in the continuous animal cell line. As the object of themethods of the invention is the generation of the animal circular ssDNAvirus or parts thereof, the methods of the invention generally comprisea further step (c), wherein the animal circular ssDNA virus is isolatedfrom the medium and/or infected cells of the continuous animal cellline.

Depending on the tools used to ensure the reduction ofendosomal-lysosomal system acidification, this can be ensured before,during and/or after the inoculation with the circular ssDNA virus.

In one embodiment of the methods of the invention, the continuous animalcell line is cultivated in the presence of an inhibitor ofendosomal-lysosomal system acidification, more particularly, alysosomotropic agent capable of reducing endosomal-lysosomal systemacidification. Most particularly, the use of ammonium chloride,chloroquine diphosphate and monensin as lysosomotropic agents areenvisaged.

In further particular embodiments, the methods of the invention comprisethe steps of: (a) inoculating cells of a continuous animal cell line inculture medium with a circular ssDNA virus and, (b) cultivating theinoculated continuous animal cell line in the presence of an agentcapable of inhibiting endosomal-lysosomal system acidification.

Yet a further aspect of the present invention relates to the use of boththe presence of interferon and the effect of endosomal-lysosomal systemacidification for improving methods for the cultivation of animalcircular ssDNA virus in an animal cell line.

Accordingly, methods are provided wherein a continuous animal cell line,prior to, during or after inoculation, is cultivated in the presence ofInterferon and simultaneously or sequentially reduction ofendosomal-lysosomal system acidification is ensured.

In one embodiment of this aspect of the invention, methods are providedcomprising the steps of (a) inoculating cells of a continuous animalcell line in culture medium with a circular ssDNA virus, (a′)administering an exogenous interferon or an agent which induces theendogenous production of an interferon by the cells of the cell lineand, (b) cultivating the continuous animal cell line in the presence ofan agent capable or inhibiting endosomal-lysosomal system acidification,whereby the steps are not necessarily in that order. Where the steps ofthe method are in that order, the methods of the invention mayoptionally comprise, between steps (a′) and (b) a change of cultivationmedium.

According to particular embodiments the methods according to thedifferent aspects of the invention are used for cultivating virusesbelonging to the group of Circoviruses, most particularly PorcineCircovirus 2 (PCV2).

According to further particular embodiments of the methods of theinvention, the cell line use in the methods of the invention is annon-human animal cell line, more particularly a porcine cell line, suchas, but not limited to PK-15, ST, SK or 3D4/31. In particularembodiments, the cell line is an epithelial cell line.

In particular embodiments, the methods of the invention encompassesensuring the presence of interferon in the medium of the cell lines.This can be achieved either by contacting the cell line withinterferons, e.g. by adding one or more exogenous interferons, such asinterferon-alpha or interferon-gamma to the medium. According toparticular embodiments, the one or more interferons are added to theculture medium at a concentration of at least 2 U/ml medium. Addition ofinterferons to the medium can be performed before, during or afterinoculation of the cell line with the animal circular ssDNA virus.

According to further embodiments of the methods of the present inventionwhich encompass ensuring the presence of interferon in the medium of thecell lines, this is achieved by the endogenous production of interferonsby the continuous cell line. In these embodiments, a continuous cellline capable of producing interferons is used. The production ofinterferons can be inherent to the animal cell line used or can be theresult of transfection with a polynucleotide which ensures interferonproduction, either constitutively or by way of an inducible promoterwhereby after transfection, stimulation of the inducible promoter by anagent results in interferon production by the cell line. Thus, accordingto these embodiments a transgenic cell line, i.e. a cell line comprisinga foreign DNA which ensures production of interferon, such as a foreignDNA encoding an interferon, is used in the methods of the invention.

According to another aspect of the invention cultivation media areprovided which comprise both animal circular ssDNA virus and at least 2U/ml interferon and/or an agent capable of inhibitingendosomal-lysosomal system acidification. According to the presentinvention such media can be used for the development of a vaccine.

Thus, according to yet another aspect of the invention methods areprovided for producing a vaccine. The methods of the invention allow amore cost-efficient way of producing animal circular ssDNA virus whichcan further be processed into a vaccine. In one embodiment, the methodfor producing a vaccine will comprise the steps of inoculating acontinuous cell line with a circular ssDNA virus and ensuring that themedium of the continuous cell line comprises interferon, moreparticularly at a concentration of at least 2 U/ml. In anotherembodiment, the methods for producing a vaccine will comprise the stepsof inoculating cells of a continuous animal cell line in a culturemedium with the circular ssDNA virus, thereby ensuring, prior to and/orinoculation, the reduction/inhibition of endosomal-lysosomal systemacidification in the continuous animal cell line. In yet a furtherembodiment, the methods for producing a vaccine will comprise the stepsof inoculating cells of a continuous animal cell line in a culturemedium with the circular ssDNA virus thereby ensuring both that themedium of the continuous cell line comprises interferon and thatendosomal-lysosomal system acidification in the continuous animal cellline is reduced. The methods according to this aspect of the inventionfurther comprise the steps of allowing the ssDNA virus to replicate inthe continuous animal cell line; and obtaining the circular ssDNA virusor components thereof from the continuous animal cell line or saidmedium.

In the methods according to this aspect of the invention, the step ofensuring that the medium of the continuous cell line comprisesinterferon is in particular embodiments ensured by adding an exogenousinterferon to the medium of said continuous cell line or adding an agentwhich ensures the endogenous production of an interferon by the cellline; a further optional step comprises allowing said ssDNA virus toreplicate in said continuous animal cell line; in further steps of themethod of the present invention, the circular ssDNA virus or componentsthereof are isolated from the continuous cell line and/or the medium ofthe cell line and used in the development of a vaccine.

In the methods according to this aspect of the invention, the step ofensuring the reduction of endosomal/lysosomal system acidification ofthe continuous animal cell line is, in particular embodiments ensured byaddition of lysosomotropic endosomal/lysosomal acidification inhibitorsto the cultivation medium. Most particularly, lysosomotropicendosomal/lysosomal acidification inhibitors are used.

According to yet another aspect the invention provides in vitro methodsfor determining the infection of a animal or a cell culture by an animalcircular ssDNA virus. Such methods involve increasing the amount ofvirus before detection. Different embodiments of this method areenvisaged within the context of the invention. In a particularembodiment the amount of virus in the sample is increased beforedetection by using interferon. In other embodiments, this increase isensured by ensuring that endosomal-lysosomal system acidification isreduced. In a further embodiment, the increase in virus titre is ensuredby the combined use of interferon and reduction of endosomal-lysosomalsystem acidification.

According to one embodiment of this aspect of the invention, methods areprovided whereby the amount of virus is directly increased in the sample(or a fraction thereof) of the animal or the culture. This method isparticularly suited for samples of animals and cell cultures thatcomprise cells, more particularly comprising cells which are susceptibleto infection by the circular ssDNA virus (such as, but not limited toblood or ascites samples or cell-comprising culture samples). Typicallyaccording to this embodiment, the method can comprise one or more stepscomprising adding interferon or an agent capable of inducing interferonto a cell-containing sample (or a fraction thereof) of said animal orcell culture and/or reducing endosomal-lysosomal system acidification inthe continuous animal cell line. The methods further comprise the stepsof allowing the replication of the circular ssDNA virus in the sample,and detecting the presence of the circular ssDNA virus in the sample.

Alternatively, the amount of virus in the sample is increasedindirectly, through the intermediate of a cell line susceptible toinfection by the animal circular ssDNA virus. This method can also beused to detect the presence of circular ssDNA virus in a sample whichdoes not comprise cells, more particularly does not comprise cellssusceptible to infection by the circular ssDNA virus (eg. supernatant orserum). Typically, according to this embodiment, the method can comprisethe steps of adding the sample (or fraction thereof) for which thedetection is to be performed to a culture of cells susceptible toinfection by circular ssDNA virus (or visa versa). The titre of thevirus is then increased e.g. by adding interferon to the latter mixture(comprising the culture of susceptible cells and the sample) and/orendosomal-lysosomal system acidification inhibitors and detecting of thepresence of ssDNA virus in the medium of the culture of cells.Alternatively, the susceptible cells are cells which produce interferoneither naturally or as a result of genetic modification. Additionally oralternatively, the susceptible cells are cells in which theendosomal-lysosomal system acidification is reduced as a result of aselection and/or genetic modification.

In the above-described methods, detection of animal circular ssDNA virusis indicative of the infection of the sample by the animal circularssDNA virus.

The above methods represent an improvement over current detectionmethods of circular ssDNA virus in animal and cell culture samples, asthe sensitivity is increased. Thus the present invention providesimprovements of viral detection methods whereby the increase insensitivity of at least ×2 is observed, compared to detection in theabsence of IFN and reduction of endosomal-lysosomal systemacidification.

In particular embodiments of the methods of the present invention, theinterferon used is IFN-alpha and/or IFN-gamma.

According to yet another aspect of the present invention, methods areprovided to improve the immune response in an animal to a vaccineagainst an animal circular ssDNA virus. In particular embodiment thesemethods comprise administration of interferon to the animal, wherein theinterferon ensures the improved immune response to said vaccine. Inanother embodiment, these methods comprise administration ofendosomal-lysosomal system acidification inhibitors to the animal,resulting in an improved immune response.

Such methods can additionally comprise the step of administering avaccine against an animal circular ssDNA virus to the animal, wherebythe administration of interferon and/or endosomal-lysosomal systemacidification inhibitors can occur sequentially to (shortly, mostparticularly within 24 hrs, before or after) or simultaneously with theadministration of the vaccine. Such methods can optionally furthercomprise identifying an animal in need of such an improved immuneresponse, either before or after administration of the vaccine thereto.

Thus the present invention provides the use of one or more interferonsand/or endosomal-lysosomal system acidification inhibitors in themanufacture of a medicament for the improvement of the immune responseto the vaccination with an attenuated animal circular ssDNA virus. Aspecific embodiment of the invention the animal circular ssDNA virus isa Circovirus.

According to this aspect of the invention, vaccines are providedcomprising both an attenuated animal circular ssDNA virus and interferonand/or endosomal-lysosomal system acidification inhibitors.Alternatively, kits for vaccination are provided which comprise a) acomposition comprising an attenuated animal circular ssDNA virus vaccineand b) an interferon and/or one or more endosomal-lysosomal systemacidification inhibitors for simultaneous or sequential administrationto an animal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the in vitro cultivation of animalcircular ssDNA viruses. The term “animal circular ssDNA virus” is usedto refer to a subgroup of animal single strand DNA (ssDNA) viruses,which infect eukaryotic non-plant hosts, and which have a circulargenome. Thus, the animal circular ssDNA viruses are to be distinguishedfrom ssDNA viruses that infect prokaryotes (i.e. Microviridae andInoviridae) and from ssDNA viruses that infect plants (i.e.Geminiviridae and Nanoviridae). At the same time they are to bedistinguished from linear ssDNA viruses that infect non-plant eukaryotes(i.e. Parvoviridiae). In the present invention “non-plant” and “animal”will be used as synonyms, whereby “animal” will include human, unlessspecified as “non-human”.

The group of circular animal ssDNA viruses encompasses both theAnelloviruses and the Circoviridae. Whether the taxonomic group of theAnelloviruses should be placed within or next to the group ofCircoviridae has not yet been completely established. Anellovirusmembers are at present not yet included in the official classificationof the ICTV (International Committee on Taxonomy of Viruses) [seeBiagini (2004) Vet. Microbiol. 98, 95-2004].

The Circoviridae represents a taxonomic group which comprises bothCircovirus and Gyrovirus. Examples of Circovirus are Psittacine Beak andFeather Disease Virus, Bovine circovirus, Canary circovirus, Columbidcircovirus, Goose circovirus, Mulard duck circovirus, Muscovy duckcircovirus, and Porcine circovirus (Porcine circovirus 1 (PCV-1) andPorcine circovirus 2 (PCV2)).

“PCV2” or Porcine circovirus 2, is a very small virus with a relativelysimple structure. The circular ambisense 1.7 Kb genome codes for twomajor proteins. On the viral strand, open reading frame (ORF) 2 codesfor the capsid protein and on the complementary strand, ORF1 codes fortwo non-structural proteins Rep and Rep′ which form a complex that isinvolved in the replication of the genome. Besides these three viralproteins, no others have been characterised to this date. The fact thatPCV2 does not code for a viral DNA-polymerase, makes it dependent ofcellular enzymes to complete its infectious cycle. Many differentstrains, isolated from PMWS-affected pigs, healthy pigs or abortedfoetuses have been characterized.

The taxonomic group of the Anellovirus comprises the SEN virus, theSentinel virus, the ITV-like mini virus and the TT virus. Differenttypes of TT virus have been described including TT virus genotype 6, TTvirus group, TTV-like virus DXL1 and TTV-like virus DXL2.

In the context of the present invention, the term ‘virus’ (such as whenreferring to an animal circular ssDNA virus can be used to refer toeither or both wild-type isolates and/or spontaneously or purposefullymutated laboratory strains. According to a particular embodiment, avirus is an attenuated virus.

The present invention relates to the cultivation of animal circularssDNA virus in an animal cell line, especially a continuous cell line.The animal cell lines envisaged within the context of the presentinvention preferably encompass any cell line that can be passagedmultiple times (e.g. at least 10 times) in vitro and which can beinfected by a circular animal ssDNA virus. The suitability of a cellline for use in the context of the present invention can be investigatedby assaying the presence of circular animal ssDNA viral proteins(immunostaining) or circular animal ssDNA viral DNA (hybridisation orPCR amplification) after infection of said cell line with an animalcircular ssDNA virus. The term cell line in the context of the presentinvention encompasses both transfected and non-transfected cell lines.

Inoculation or infection of a susceptible animal cell line by an animalcircular ssDNA virus can be achieved by routine methods. Typicallyinoculation is performed as described herein using a strain of circularssDNA virus at a multiplicity of infection ranging from about 0.01 toabout 1. The virus is usually diluted in MEM and inoculated on the cellcultures by incubating cells and virus for 1 hour at 37° C. After 1hour, new culture medium is added.

A particular embodiment of the present invention relates to thecultivation of PCV2 in continuous animal cell lines in vitro. Accordingto one embodiment of the invention, the cell lines are porcine celllines. PCV2 is able to replicate in most porcine cell lines in vitro.Most particularly, the cell lines envisaged in the context of thepresent invention are immortalised porcine cell lines such as, but notlimited to the porcine kidney epithelial cell lines PK-15 and SK, themonomyeloid cell line 3D4/31 and the testicular cell line ST. PCV2 isalso able to replicate to a lesser extent in CHO cells (Chinese hamsterovaries). A number of non-porcine cell lines are resistant toPCV2-infection: MARC-145, MDBK, RK-13, EEL). Additionally oralternatively, particular embodiments of the methods of the inventionmake use of an animal cell line which is an epithelial cell line, i.e. acell line of cells of epithelial

Another embodiment of the present invention relates to the cultivationof TTvirus in animal cell lines in vitro. In the context of thisembodiment continuous cell lines susceptible to infection with TTvirusare envisaged. Such cells lines can be, but are not limited to celllines of human or primate origin, such as human or primate kidneycarcinoma cell lines.

A particular embodiment of the present invention relates to thecultivation of PCV2 in continuous animal cell lines, for the productionof vaccines, more particularly vaccines for use in pigs. When referringto “Pigs” in the context of the present invention, reference is made toa member of the Suidae, more particularly, any race or strain of Susscrofa domestica. It includes free living and domesticated pigs. It alsoincludes to pigs, which underwent a special regimen such as SPF(Specific Pathogen Free) pigs, or gnotobiotic pigs (gnotobiotic piglets(caesarian-derived, colostrum-deprived, raised in a germ-freeenvironment).

The present invention refers to a method for the in vitro cultivation ofan animal circular ssDNA virus. According to a particular embodiment thecircular ssDNA virus is a member of the Circoviridae or is anAnnelovirus. In a particular embodiment of the present invention, thecircular ssDNA virus is a Circovirus, most particularly PCV2. Moreparticularly, the method of the invention is demonstrated for thecultivation of PCV2 strain Stoon-1010. This strain was isolated from thefirst described case of PMWS and can therefore be considered to be thereference strain.

According to a first aspect of the present invention improvedcultivation of the circular ssDNA virus (i.e. increased virus titre) isobtained in vitro by infection of a continuous animal cell line with thecircular ssDNA virus in the presence of an interferon. Interferonsuseful in the context of the present invention include Type I and TypeII interferons. According to a particular embodiment of the presentinvention, IFN-gamma is used. Apart from wild type interferons (isolatedfrom mammalian or bacterial cells or recombinant), the invention canalso be performed with modified versions (mutated and/or truncatedversions) of an interferon, as long as the IFN remains active in one ofthe bioassays known to the skilled person, such as, but not limited tothe one described herein for IFN-alpha.

According to a particular embodiment of the invention the methodencompasses the addition of one or more exogenous interferons to thecell culture. The term ‘exogenous’ in this context means that theinterferon(s) is(are) not produced by the cell line itself but addeddirectly or indirectly to the medium.

The concentration of interferons added to the culture of animal circularssDNA viruses according to the present invention is a concentration ofat least 2 U/ml, particularly at least 50 U/ml, more particularly atleast 100 U/ml. Typical concentrations of IFN-gamma added to large scaleproduction units will be around 250-500 U/ml, but most likely evenhigher concentrations will further increase virus titres.

The addition of exogenous interferons can be achieved in different ways.According to a particular embodiment of the invention, the cytokine isadded in purified form, e.g. from a cytokine stock as described above.Alternatively, however, the interferon can be present in a medium, e.g.as obtained from a cell culture which produces interferon. Typically,immunological cells are capable of producing interferons whenstimulated. An exemplary source of IFN may be the medium of PBMCs, or ofany specific cell type producing a type of IFN such as but not limitedto leukocytes, fibroblasts, epithelial cells, macrophages, lymphocytes,plasmacytoid dendritic cells, and bacterial cells. Compounds which areknown to induce IFN production in certain cell types and which can beused in the context of the present invention include, but are notlimited to concanavalin A, 12-O-tetradecanoyl-phorbol-13-acetate, IL-2,Poly I:C. Alternatively, interferon production can be induced in anumber of cell types by infection with a micro-organism or foreignagent, e.g. of viral, bacterial or parasitic origin or contacting thecells with non-infectious proteins. Methods to induce IFN production incultures by viruses have been described, e.g. in U.S. Pat. No.3,951,740. Such a virus can be a second animal virus, such as porcinereproductive and respiratory syndrome virus (PRRSV) or porcineparvovirus (PPV). Alternatively, e.g. in the case of human cells, theother virus can be an animal virus such as Hepatitis C virus or HIV.

According to a further embodiment of the present invention the methodfor cultivating animal circular ssDNA virus encompasses ensuring theproduction of interferons by the cell line itself, i.e. the productionof endogenous interferons.

A limited number of continuous cell lines are capable of producinginterferons, such as, but not limited to lymphoblastoid cell lines (e.g.Namalva cell lines producing “Namalva interferon”). Most continuousanimal cell lines however do not naturally produce interferons.Nevertheless, the present invention also envisages cell lines capable ofproducing interferon as a result of transfection. Cell lines capable ofproducing IFN as a result of genetic manipulation are generally referredto herein as transgenic cell lines. Such a production of one or moreinterferons by transfected cell lines can be either constitutive orinducible. Methods of transfecting cell lines suitable in the context ofthe present invention, such as, but not limited to the PK15 cell line,are known in the art. The cloning and expression of porcine interferonalpha and gamma is described in Xia et al (2005) Vet ImmunolImmunopathol. 104, 81-89. Alternatively, a wide variety of vectors forthe recombinant expression of genes in eukaryotic cells are availablefrom e.g. Clontech, InVitrogen, Stratagene. DNA sequences encodingporcine interferon gamma [NM_(—)213948] and interferon alpha[NM_(—)214393] are deposited in Genbank.

Protocols for the transfection of cell are described in e.g. CellBiology: A Laboratory Handbook (1998) Ed. J. Celis, Academic Press, orare available from the manufacturers of transfecting agents (eg Fugene(Roche Diagnostics) or electroporation apparatus (e.g. BioRad).

According to this aspect of the invention endogenous production ofinterferon by the cell line is ensured by transfection of the cell lineand, in the case of inducible promoters, induction of the inducibleinterferon production by a compound which is capable of activating theinducible promoter.

The amount of interferon used or observed in the context of the presentinvention will be referred to as a concentration (i.e. amount/ml). Aconcentration can be expressed in weight (mg/ml) or molarity (M) or byactivity (Units/ml).

Assays for determining activity in Units/ml are known in the art. Asthere is no standard for porcine interferon, the units of porcineinterferons are usually determined with respect to the internationalreference standard for the corresponding human interferon. For instancethe units of porcine interferon alpha is determined with respect to theactivity of human leukocyte interferon (Ga23-902-530) provided by theNational Institutes of Health [see Pestka, S. (1986) Methods inEnzymology 119, 14-23]. The activity of porcine IFN-alpha can bedetermined using the cytopathic effect inhibition assay as described byRubinstein et al S. [(1981) J. Virol. 37, 755-758] and Famillett et al.[(1981) Methods in Enzymology 78, 387-394]. In such an antiviral assayabout 1 unit/ml of interferon is the quantity necessary to produce acytopathic effect of 50%.

In the context of the present invention, interferon concentrations willrefer to the exogenously added interferon as calculated over the amountof cell culture medium. Alternatively, when IFN is produced endogenouslyby the cell line according to a particular embodiment of the invention,IFN concentrations in the medium will reflect the amount of interferonproduced by the cells which are present in the cell culture medium. Theactivity and concentration of interferon(s) can be assayed byquantitative measurements (e.g. ELISA) or by qualitative measurements(bioassays).

Thus, typically the medium of the continuous cell lines used to produceanimal circular ssDNA viruses according to the present invention willcontain one or more interferons at a concentration of at least 2 U/ml,more particularly at least 20 U/ml, even more particularly at least 100U/ml, most particularly 500 U/ml or more.

Where the interferon is exogenously added or endogenous production canbe controlled (e.g. use of an inducible promoter), according toparticular embodiments of the methods of the present invention, the timeperiods envisaged for contacting the cells with interferon will bebetween 30 minutes and 48 hrs or more. It will be understood that thetime period is determined at least in part by the concentration of IFNused or envisaged to be produced by the cells. More particularly, a timeperiod of 24 hrs incubation in the presence of 100-500 U/ml interferonis envisaged.

According to another aspect of the invention, improved cultivation ofthe circular ssDNA virus (i.e. increased virus titre) is obtained invitro by infection of a continuous animal cell line with the circularssDNA virus and inhibiting (with the aim of at least reducing)endosomal-lysosomal system acidification in the cells. Theendosomal-lysosomal system consists of membrane bound organelles thatfunctions in the cellular housekeeping by internalization, sorting, andbreakdown of macromolecules (Mellman, I. 1996. Annu. Rev. Cell Dev.Biol. 12, 575-625). The endosomal-lysosomal system is composed ofprimary endocytic vesicles, early endosomes, late endosomes, andlysosomes. The endosomal-lysosomal system is characterized by gradualacidification of its vesicles as they mature from early endosomes intolysosomes. It has been found that reduction of endosomal-lysosomalsystem acidification increases susceptibility of certain cells to PCV2infection.

Accordingly, the present invention envisages methods wherebyendosomal-lysosomal system acidification is reduced or inhibited.Endosomal-lysosomal system acidification can be inhibited bylysosomotropic agents. The term ‘lysosomotropic’ as used hereindesignates all substances that are taken up selectively into lysosomesirrespective of their chemical structure or mechanism of uptake (De Duveet al., 1974. Biochem. Pharmacol. 23:2495-2531). The pH of endosomaland/or lysosomal vesicles can be measured by methods known to theskilled person (such as, but not limited to described inter alia byMaxfield et al. 1982, J. Cell Biol. Vol 95:676-681; Ohkuma and Poole1987, Proc. Natl Acad Sci USA 75:3327-3331). In particular embodimentsthe reduction of acidification envisaged corresponds to an increase ofpH of at least 0.5 units, more particularly 1 unit or more.

According to one embodiment, a lysosomotropic agent is used, moreparticularly a lysosomotropic acidification-inhibiting agent, i.e. alysosomotropic agent capable of changing the acidic pH of the lysosomesand/or other vesicles within the endosomal-lysosomal system.Lysosomotropic agents, such as chloroquine diphosphate, ammoniumchloride and monensin raise pH within lysosomes, thereby also resultingin an increased pH. It has been found in the present invention thatreduction of endosomal-lysosomal system acidification by treatment ofcells with lysosomotropic agents capable of raising the pH within theendosomal-lysosomal system increases the susceptibility of cells to PCV2infection. Particularly, suitable lysosomotropic agents for use in themethods of the present invention thus include, but are not limited to,ammonium chloride, chloroquine diphosphate and monensin.

Further examples of suitable inhibitors of endosomal-lysosomalacidification include tamoxifen (Nihal et al., 1999. Proc Natl Acad SciUSA 96(8):4432-7), bafilomycins and concanamycins (Drose & Altendorf.1997. J. Exp. Biol. 200:1-8), nigericin,N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl-methylamine)dihydrochloride(DAMP) (Anderson et al., 1984. Proc Natl Acad Sci USA 81:4838-4842),primaquine (DeDuve et al., 1974, Biochem Pharmacol 23:2495-2531),methylamine, Destruxin B (Muori et al., 1994. Biochem Biophys Res Commun205:1358-1365).

According to another embodiment, endosomal acidification is reduced orinhibited by other means, such as, but not limited to, exposure tocadmium or pyocyanin (produced by Pseudomonas aeruginosa) or otheragents that directly inhibit V-type H+-ATPase (proton pump; V-ATPase),e.g. bafilomycins and concanamycins, and/or inhibit intracellularvesicle trafficking within the endosomal-lysosomal system; specificantibiotics such as duramycin and azithromycin capable of inhibitingacidification of the endosomal-lysosomal system; Amphotericin B, anantifungal drug, which blocks the fusion between endosomes and/or thefusion between endosomes and lysosomes; infection with Rhodococcus equiand Francisella tularensis, bacteria that have been described to survivea phagolysosomal environment by suppressing acidification of thephagolysosome (Clemens et al., 2004. Infect Immun. 72:3204-17; Toyookaet al., 2005. J Med Microbiol. 54:1007-15); finally the infection ofcell lines carrying genetic modifications resulting in loss of functionof proteins involved in endosome acidification machinery is alsoenvisaged, including cell lines which naturally or via selection havedefective endosome acidification (such as CHO mutants DTG 1-5-4 and DTF1-5-1 (Robbins et al. 1983, J. Cell Biol. 99:1296-1308)).

Accordingly, according to this aspect of the invention, methods areprovided for the in vitro cultivation of an animal circular ssDNA viruscomprising the step of inoculating cells of a continuous animal cellline in a culture medium with said circular ssDNA virus, whileinhibiting endosomal-lysosomal acidification. In a particularembodiment, the reduction of endosomal-lysosomal acidification isensured by cultivating the cells in the presence of a pH stabilizingagent. More particularly, the pH stabilizing agent is a lysosomotropicagent capable of inhibiting acidification within lysosomes.

Where a stabilizing agent is used according to the methods of thepresent invention, incubation of the continuous cell line with thestabilizing agent can be performed prior to, simultaneously with, orsubsequent to PCV2 inoculation of the cells. According to a particularembodiment, the cells are treated with one or more stabilizing agentsafter PCV2 inoculation.

The amount of stabilizing agent used in the methods of the invention isdetermined by the nature and characteristics of the agent used. Wherethe use of lysosomotropic agents is envisaged, the amount of agent istypically between 0.1 μM and 200 mM. The present invention demonstratesthat significant enhancement of PCV2 infection can be obtained by using25 mM ammonium chloride, 125 μM chloroquine diphosphate and/or 6 μMmonensin. Typically incubation with stabilizing agents takes place for aperiod between 30 min and 2-5 days. Most particularly an incubation timeof 24 hrs with the agents of the invention is envisaged.

Where other methods of inhibiting endosomal-lysosomal systemacidification are used, other concentrations and/or time periods ofincubation may be appropriate. These can be easily optimized by theskilled person by optimization experiments as performed for thelysosomotropic agents described herein.

According to a further aspect of the invention, improved cultivation ofthe circular ssDNA virus (i.e. increased virus titre) is obtained invitro by infection of a continuous animal cell line with the circularssDNA virus in the presence of an interferon and reduction ofendosomal-lysosomal system acidification in the cells. According to thisaspect, the effect of endosomal-lysosomal system acidification reductionand the presence of an interferon on the susceptibility of cells to PCV2infection is combined. More particularly the combined use ofendosomal-lysosomal system acidification inhibitors and IFN generates asynergistic effect on PCV2 production.

Accordingly, according to this aspect of the invention, methods areprovided for the in vitro cultivation of an animal circular ssDNA viruscomprising the step of inoculating cells of a continuous animal cellline in a culture medium with said circular ssDNA virus, in the presenceof interferon, as described hereinabove, and cultivating the continuousanimal cell line while inhibiting endosomal-lysosomal systemacidification. Generally, the methods according to this aspect of theinvention involve a combination of the methods of cultivation in thepresence of IFN and the methods of cultivation wherebyendosomal-lysosomal system acidification is inhibited as described inthe sections above. According to one embodiment the addition ofinterferon and the reduction of endosomal-lysosomal system acidificationis performed simultaneously. Alternatively, the addition of interferonand the reduction of endosomal-lysosomal system acidification can beperformed in subsequent steps, either in the same medium or withintermediate removal of the cultivation medium. According to aparticular embodiment of the present invention, the cells are contactedwith interferon prior to the inoculation with PCV2, whereafter thecultivation medium is changed and an agent ensuring inhibition ofendosomal acidification is added to the new medium (with or withoutinterferon) for further cultivation. Where presence of IFN is ensured byendogenous production of IFN by the cells, endosomal acidificationinhibitors can be added prior to or shortly after inoculation of thecells with PCV2.

According to one aspect, the present invention provides methods for thecultivation of viruses, whereby increased virus titres are obtained,which is of interest e.g. in the production of vaccines. The averagetitre of virus in a medium of infected cells is between 3.4 and 4.5log₁₀ TCID₅₀/ml. Using the methods of the present invention, theconcentration of an animal circular ssDNA virus in an undiluted culturemedium, can raise up to a titre of 4.8 to 5.7 log₁₀ TCID₅₀/ml or more.Thus, the present invention relates to an improvement of the cultivationof animal circular ssDNA virus which ensures an increase in titre ofapproximately 1.3 log₁₀ TCID50/ml or more. It is demonstrated hereinthat using the methods of the invention, titres corresponding to titresrepresentative of industrial production can be obtained.

The production of virus-containing cell cultures according to thepresent invention can be carried out in different scales, such as inflasks, roller bottles or bioreactors. The media used for thecultivation of the cells to be infected are known to the skilled personand will comprise the standard nutrients required for cell viability butmay also comprise additional nutrients dependent on the cell type.Optionally, the medium can be protein-free. Depending on the cell typethe cells can be cultured in suspension or on a substrate.

Thus, one aspect of the invention relates to circular animal ssDNAvirus, which can be isolated from a medium and/or infected cells aftercultivation according to the present invention. The isolated virus canbe used to obtain one or more of the viral proteins or to isolate theviral DNA therefrom.

The purification and isolation of circular animal ssDNA virus is knownby the skilled person and is described for example by Meehan et al.(1998) J. Gen. Virol. 79, 2171-2179. Protection of swine againstpost-weaning multi-systemic wasting syndrome by PCV2 proteins has beendemonstrated (Blanchard et al. (2003) Vaccine 21, 4565-4575).

Alternatively, the isolated virus can be used as such in the productionof an attenuated or inactivated virus for vaccination. Attenuation ofvirus strains for use in vaccination is performed by different methods,including repeated passaging on cells, particularly on cell lines, suchas PK/15 or by activation of virus-associated endonuclease (Schodelleret al. J. Gen. Virol., 65, 1567-1573). Inactivation of a virus can beachieved by using chemical methods, e.g. by exposing the antigen to achemical agent such as formaldehyde (formalin), paraformaldehyde,beta.-propiolactone or ethyleneimine or its derivatives (Larghi et al.(1980) J. Clin Microbiol 11, 120-122); US patent application2002/0146432), or by UV-irradiation.

Thus, one aspect of the present invention relates to a method for thepreparation of a vaccine against a circular animal ssDNA virus, whichencompasses the cultivation of the virus in accordance with the methodsof the present invention. A vaccine according to the present inventioncan comprise either an immunogenic agent or a compound which, uponintroduction into the host, ensures the production of an immunogenicagent. Thus a vaccine can comprise DNA, RNA, or protein material, orboth, including the complete virus.

In a particular embodiment the vaccine is a vaccine for the preventionof postweaning multisystemic wasting syndrome (PMWS) in pigs caused byPCV2 strains such as strain Stoon-1010. In another particular embodimentthe vaccine is a vaccine for the prevention of the abortion of pigscaused by the abortion-associated PCV2 strain 1121. In anotherparticular embodiment the vaccine is a vaccine for the prevention ofhepatitis-like disorders caused by Anelloviruses such as TT virus.

Besides the immunogenic agent or the compound which ensures theproduction of the immunogenic agent in vivo, vaccines generally comprisea vehicle or diluent acceptable from the veterinary point of view,optionally an adjuvant acceptable from the veterinary point of view, aswell as optionally a freeze-drying stabilizer. When comprisingattenuated virus particles, vaccines will generally comprise from10^(3.0) to 10^(6.0) TCID50 (50% tissue culture infective dose).Inactivated vaccines can be supplemented with adjuvant, advantageouslyby being provided in the form of emulsions, for example water-in-oil oroil-in-water, according to techniques well known to persons skilled inthe art. It will be possible for the adjuvant character to also comefrom the incorporation of a customary adjuvant compound into the activeingredient. Among the adjuvants which may be used, there may bementioned by way of example aluminium hydroxide, the saponines (e.g.Quillaja saponin or Quil A; see Vaccine Design, The Subunit and AdjuvantApproach, 1995, edited by Michael F. Powel and Mark J. Newman, PlennumPress, New-York and London, p. 210), Avridine® (Vaccine Design p. 148),DDA (Dimethyldioctadecyl-ammonium bromide, Vaccine Design p. 157),Polyphosphazene (Vaccine Design p. 204), or alternatively oil-in-wateremulsions based on mineral oil, squalene (e.g. SPT emulsion, VaccineDesign p. 147), squalene (e.g. MF59, Vaccine Design p. 183), orwater-in-oil emulsions based on metabolizable oil (preferably accordingto WO-A-94 20071) as well as the emulsions described in U.S. Pat. No.5,422,109 or those described in WO-A-9416681. It is also possible tochoose combinations of adjuvants, for example Avridine® or DDA combinedwith an emulsion. As freeze-drying stabilizer, there may be mentioned byway of example SPGA (Bovarnik et al., J. Bacteriology 59, 509),carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran orglucose, proteins such as albumin or casein, derivatives of thesecompounds, or buffers such as alkali metal phosphates.

Another aspect of the present invention relates to the provision of amethod for diagnosing an infection of animal circular ssDNA virus in asample, whereby the sensitivity of the detection is increased. Thesemethods are of interest in the detection of postweaning multisystemicwasting syndrome (PMWS) or susceptibility thereto, as pigs with highPCV2 replication have been shown to be more likely to develop PMWS thanpigs with low PCV2 replication (Segalés and Domingo, 2002, Vet Q. 2002September; 24(3):109-24). The sample used in the detection methodsaccording to this aspect of the present invention. Such a sample can beeither a cell-containing sample (which as used herein refers to a samplecomprising cells which are susceptible to animal circular ssDNA virus)or a sample which itself does not comprise cells which are susceptibleto infection with circular ssDNA virus (e.g. supernatant of a cellculture). Typically, the sample will be either a cell-containing or noncell-containing sample of an animal or a cell culture. The detection caneither be performed on the sample or on a fraction thereof, but forsimplification purposes the methods of the invention will generallyrefer to the sample. The increase in sensitivity of detection isobtained by increasing the amount of virus before detection usinginterferon and/or inhibiting endosomal-lysosomal system acidification asdescribed herein. This increase in amount of virus can be achieved inthe sample directly, whereby detection of the ssDNA virus can also beperformed on the sample directly. Alternatively, the increase in amountof virus is achieved after addition of the sample to a cell culturesusceptible to animal circular ssDNA virus infection and ensuring themedium of the cell culture contains or is contacted with interferonand/or conditions inhibiting endosomal/lysosomal system acidification.This can be ensured by addition of interferon or an interferon inducingagent to the medium of the cell culture or by ensuring that the cellculture endogenously produces interferon. Additionally or alternatively,this is ensured by addition of one or more endosomal/lysosomalacidification inhibitors to the cell culture or by ensuring that thecell culture endogenously generates an increased pH in the lysosomes orother organelles within the endosomal-lysosomal system (e.g. as a resultof genetic modification of a gene involved in endosomal/lysosomalacidification). When a cell culture susceptible to animal circular ssDNAvirus infection is used, detection of the presence of ssDNA virus isperformed on the cells or the medium of the cell culture.

Thus, according to a first embodiment of this aspect of the invention,the sample is e.g. a cell-comprising sample of an animal, such as ablood sample. Addition of an IFN, or an agent capable of inducing IFNproduction (by the cells in the sample) to the sample of the animal, isused to obtain an increase in virus-titre in the sample such that thesensitivity of the detection of the circular ssDNA virus in the sampleis increased. Agents capable of inducing IFN in e.g. blood cells aredescribed herein. Additionally or alternatively, addition of an agentcapable of inhibiting endosomal/lysosomal acidification or an agentwhich indirectly ensures reduction of acidification in theendosomal-lysosomal system is used to ensure in an increase invirus-titre in the sample, similarly resulting in an increasedsensitivity of detection. Where both interferon and reduction ofendosomal/lysosomal acidification are used, a synergistic effect onsensitivity is obtained.

According to another embodiment of this aspect of the invention, thesample (which can be a sample which does not contain cells, such asserum or cell culture supernatant) is contacted with a cell culturewhich is susceptible to infection by a circular ssDNA virus, to whichthe interferon and/or agent inhibiting endosomal/lysosomal acidificationis then added. Alternatively, the production of interferon by the cellculture used can be ensured, either by selecting an appropriate IFNproducing cell line or by genetic modification of a susceptible cellline. Similarly, a cell line with altered acidification of theendosomal/lysosomal system can be used. Detection of the circular ssDNAvirus is performed on the medium or the cells of the cell culture. Sucha detection can be performed both qualitatively and/or quantitativelybased on the detection of the presence of viral DNA, viral proteins orthe detection of infectious virus. Again, cultivation according to themethods of the invention will induce a higher virus titre such that thesensitivity of the detection is increased. According to this aspect ofthe invention the cell cultures susceptible to animal circular ssDNAvirus infection can be a continuous cell line or can be a primary cellculture such as a culture of PBML's.

According to yet another aspect of the invention interferon and/orinhibitors of endosomal-lysosomal system acidification are used tomediate the response of animals, more specifically pigs or humans to thevaccination with an attenuated circular ssDNA virus. Attenuated virusesare modified viruses which still replicate in the host but are no longer(or less) virulent, and can be obtained as described herein. Accordingto this aspect interferon and/or inhibitors of endosomal-lysosomalsystem acidification are used to increase the replication of attenuatedPCV2 in vivo and thus to enhance the immune response to the virus uponadministration to an animal. Administration of the interferon and/orinhibitors of endosomal-lysosomal system acidification to the animal canbe done separately (i.e. before, during or after administration of thevirus vaccine) or together with the virus. Thus one aspect of theinvention relates to a vaccine comprising both an animal circular ssDNAvirus and interferon and/or inhibitors of endosomal-lysosomal systemacidification.

The following Examples, not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figures:

FIG. 1. Influence of cytokines on number of PCV2-infected PK-15 cellsaccording to one embodiment of the invention.

-   -   Black bar: effect of cytokines administered before inoculation;        grey bar: during inoculation; white bar: post inoculation. All        results represent the mean of three independent        experiments+standard error of the mean.

FIG. 2. Light microscopic pictures of the influence of IFN-gammatreatment on the number of PCV2 positive cells in PK-15 and 3D4/31 cellsaccording to one embodiment of the invention. Pictures A and C show thenumber of PCV2 positive cells in respectively PK-15 and 3D4/31 cells.Pictures B and D show the number of PCV2 positive cells in respectivelyPK-15 and 3D4/31 cells treated with 500 U/ml IFN-gamma respectivelyafter and before inoculation. All pictures are taken at a magnificationof 100×.

FIG. 3. Influence of cytokines on number of PCV2-infected 3D4/31 cellsaccording to one embodiment of the invention.

-   -   Black bar: effect of cytokines administered before inoculation;        grey bar: during inoculation; white bar: post inoculation. All        results represent the mean of three independent        experiments+standard error of the mean.

FIG. 4. Progeny virus production in PK-15 cells treated with 500 U/mlIFNγ compared to non-treated cells according to one embodiment of theinvention. Full lines represent the titres obtained in PCV2-inoculatedPK-15 cultures supplemented with 500 U IFN-γ per ml. Dashed linesrepresent titres obtained in PK-15 cultures without IFN-γ. Lines withblack circles represent extracellular virus titres, lines with blackcubes represent intracellular titres. Lines without symbols representtotal PCV2 titres±standard error of the mean.

FIG. 5. Effect of interferon-gamma on attachment of recombinantPCV2-capsid virion-like particles to 3D4/31 cells according to oneembodiment of the invention.

-   -   Black blocks represent the number of virion-like particles        attached at different time points after inoculation tot        non-treated    -   3D4/31 cells. White blocks represent the results obtained in    -   3D4/31 cells treated with 500 l/ml IFN-gamma during incubation        of the virion-like particles with the cells. The results        represent the average number of virion-like particle attached to        10 different cells±standard deviation.

FIG. 6. Effect of naturally induced interferons on PCV2-infection inPK-15 cells according to one embodiment of the invention; Effect ofBAL-fluids of PRCV and mock-inoculated pigs (A) and supernatant of ConAand mock-stimulated PBMC's (B) on the number of PCV2-infected PK-15cells.

FIG. 7. Effect of endosomal-lysosomal system acidification inhibitorsand/or IFN-gamma on the number of PCV2-infected PK-15 cells. PK-15 cellswere pre-treated with (black bars) or without IFN-gamma (white bars) for24 hours before they were inoculated with the same dose of PCV2. AfterPCV2 inoculation, cells were treated with (ammonium chloride,chloroquine diphosphate and/or monensin) or without (control)endosomal-lysosomal system acidification inhibitors for 24 hours. Thenumber of PCV2-infected cells in all treatment combinations was countedafter 36 hours incubation and compared with the number of PCV2-infectedcells in non-treated cells.

FIG. 8. Total PCV2 virus titres in PK-15 cells treated with IFN-gammaand/or inhibitors of endosomal-lysosomal system acidification; 25 mMammonium chloride, 125 μM chloroquine diphosphate and/or 6 μM monensin.

EXAMPLES General methodology

Cells, Virus and Inoculation

PK/15 is a pig kidney epithelial cell culture derived from embryonicpigs, which is known to be uncontaminated with the porcine circovirus(PCV), pestiviruses, porcine adenoviruses and porcine parvoviruses[Allan G. et al. (1995) Vet. Microbiol. 44, 49-64].

PK-15 cells were seeded and maintained in culture medium containing5%-10% fetal bovine serum (FBS), 0.3 mg/ml glutamine, 100 U/mlpenicillin, 0.1 mg/ml streptomycin, 0.1 mg/ml kanamycin dissolved inminimal essential medium (MEM) (Gibco BRL®, Grand Island, USA) ordissolved in RPMI-1640 (GIBCO BRL®, Grand Island, USA) containing 1%nonessential amino acids (100×; GIBCO BRL®, Grand Island, USA) and 1 mMsodium pyruvate (GIBCO BRL®, Grand Island, USA). Cells were seeded at aconcentration of 150,000 cells per ml of medium. For the detection oftotal viral antigen positive cells, the cells were seeded in 96-wellmicrotiter plates (Nunc) (15,000-20,000 cells per well). To detect theexpression of specific viral antigens, cells were seeded on sterilecover slips in Leighton tubes (150,000 cells per tube) and finallyprogeny virus production assays were performed in 24-well platescontaining 15,000 cells per well.

In order to use cells, more related to the target cells in vivo, themonocytic cell line 3D4/31 was used to confirm the effect of thedifferent cytokines on the number of PCV2-infected cells. The 3D4/31cell line is a porcine monomyeloid cell lines that was establishedfollowing transfection of primary porcine alveolar macrophage cultureswith plasmid pSV3neo, carrying genes for neomycin resistance and SV40large T antigen. (Weingartl et al. (2002) J Virol Methods. 10, 203-216).These cells were maintained in a 1:1 mixture of RPMI-1640 (Invitrogen)and DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 0.3mg/ml glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.1 mg/mlkanamycin and 1% non-essential amino acids 100× (GIBCO BRL®, GrandIsland, USA). These experiments were also performed in 96-well platescontaining the same number of cells. Furthermore, to monitor the effectof IFN-gamma on the entry of PCV2 virion-like particles was determined,3D4/31 cells were seeded at 2×10⁵ cells/ml of medium onto microscopicslides mounted with an 8 well cell culture silicone chamber (VivascienceAG, Hanover). In all inoculations, the same PCV2-strain (Stoon-1010) wasused at a multiplicity of infection of 0.01. The virus was diluted inMEM and inoculated on the cell cultures by incubating cells and virusfor 1 hour at 37° C. After 1 hour, the inoculum was removed, thecultures were washed twice with MEM and new culture medium was added.

Cytokines and Neutralizing Antibodies

Porcine interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 10(IL-10), and interferon gamma (IFN-gamma) were all purchased from thesame company (R&D systems). The activity of the preparations isindicated on the leaflets supplied with the product. In case ofIFN-gamma the activity is measured in an anti-viral assay using porcinePK-15 cells infected with EMC virus. The ED50 for this effect istypically 0.015-0.045 ng/mL.

Porcine recombinant interferon alpha (IFN-alpha) was kindly provided byDr. Charley [Lefebvre F. et al. (1990) J Gen Virol. 71, 1057-1063].Tumour necrosis factor alpha (TNF-alpha) was produced in L929 cellstransfected with the pBMGNeo expression-vector containing the TNFαcoding cDNA (Von Niederhausern et al. (1993) Vet Immunol Immunopathol,38, 57-73). IL-1, IL-6, IL-10 and IFN-gamma were dissolved according tothe manufacturers' instructions in lipopolysaccharide free phosphatebuffered saline (PBS) supplemented with 0.1% bovine serum albumin(Sigma, Bornem, Belgium) to a concentration of 10 μg/ml. Subsequently,the cytokines were diluted in MEM supplemented with 10% FBS to aconcentration eight times higher than the highest final concentrationused in the assays.

IFN-alpha neutralizing antibodies (K9) were kindly provided by Dr.Charley.

Statistical Analysis

All experiments were repeated three times independently. The resultspresented herein represent the mean value obtained from these treeexperiments. The variation between different experiments is representedby the standard error of the mean (SEM). Differences were considered tobe significant when p<0.05. (p-value calculated with the Mann-Whitneytest).

Example 1 Influence of Cytokines on the Total Number of PCV2-InfectedCells

The influence of the cytokines on the infection of PCV2 in PK-15 and3D4/31 cells was determined by adding two-fold-dilution series of thecytokines to the medium of the cells before, during or after theinoculation. IL-1, IL-6, IL-10 and IFN-alpha were used in concentrationsranging from 0.25-250 Units/ml (U/ml), IFN-gamma was used inconcentrations ranging from 0.25-1000 U/ml and IL-10 was used inconcentrations from 0.13-125 U/ml. Cell cultures were either pre-treatedwith the cytokines for 24 hours before inoculation, treated during theinoculation or the cytokines were added in the medium after theinoculation. After 36 hours of incubation at 37° C. in an environmentsupplemented with 5% CO2, the cells were fixed by drying and frozen at−20° C. The plates were stained with an immuno-peroxydase monolayerassay (IPMA) as described by Sanchez et al. (2003, Vet Microbiol 95,15-25) and the number of PCV2-positive cells was counted by lightmicroscopy. In each plate a control was inoculated with an equal dose ofmock-treated PCV2 (treated with MEM without cytokines). The number ofinfected cells in this well was used as the reference and all resultswere expressed as a ratio to this reference.

Influence of Cytokines on the Total Number of PCV2-Infected PK-15 Cells

The results of the effects of dilution series of different cytokines inPK-15 cells are shown in FIG. 1. With TNF-alpha, IL-1, IL-6 and IL-10,no significant change in the number of PCV2-positive cells was observedat any concentration or for any time of treatment (before, during orafter inoculation). With both interferons (IFN-alpha and IFN-gamma) aclear effect was observed.

IFN-gamma induced a dose-dependent increase in the number ofPCV2-antigen positive cells disregarded of the time point when it wasadded to the medium of the cells (before, during of after theinoculation). The highest effect was seen with the highest concentrationtested in the experiment (1000 U/ml). When this concentration ofIFN-gamma was supplemented to the medium before inoculation, an increasein positive cell of 518±134% was observed, when it was added to themedium during inoculation, an increase of 270±57% was observed and thehighest effect was observed when it was added to the medium after theinoculation, which induced an increase of 791±105%.

The lowest concentration of IFN-gamma which induced a significantincrease of PCV2-positive cells when administered before, during orafter inoculation, were respectively 16, 2 and 2 U/ml). FIG. 2 shows apicture of light microscopic views of PK-15 cells without IFN (negativecontrols) and with 500 U/ml IFN-gamma added in the medium after theinoculation.

IFN-alpha induced a similar increase of positive cells when administeredduring or after inoculation, but it induced a significant reduction ofinfected cells when the cells were pre-treated. At the highestconcentration used (250 U/ml) an increase of 341±114% and 629±59% wasobserved when added respectively during or after the inoculation. Afterpre-treatment, a reduction of 31±6% was observed. The lowestconcentrations of IFN-alpha inducing a significant effect when addedbefore, during or after PCV2-inoculation were respectively 31, 16 and 8U/ml). The effects induced by treatment of cells with IFN-alpha could beneutralized when IFN-alpha was incubated for 1 hour at 37° C. withIFN-alpha-neutralizing antibodies, prior to incubation with the cells.

Influence of Cytokines on the Total Number of PCV2-Infected 3D4/31 Cells

In 3D4/31 cells, TNF-alpha, IL-1, IL-6 and IL-10, did not induce asignificant change in the number of PCV2-positive cells at anyconcentration or at any time of treatment (before, during of afterinoculation). Similar observation were made in these cells compared toPK-15 cells when the 3D4/31 cells were treated with IFN-alpha andIFN-gamma. IFN-gamma induced a dose-dependent increase in the number ofPCV2-positive cells when cells were treated before, during or after theinoculation. A maximal increase of respectively 806±88%, 214±16% and523±115% was observed. When cells were treated with IFN-alpha beforeinoculation, no significant changes were observed. When IFN-alpha wasadministered to the cells during or after inoculation, an increase inpositive cells of respectively 115±10% and 408±35% was detected. Theresults of these experiments are presented in FIG. 3.

Example 2 Influence of IFN-Gamma on the Production of PCV2

Since IFN-gamma increased the number of PCV2-positive PK-15 cellsindependent of the time when it was added to the medium of the cells,this cytokine was selected to investigate the effect in the productionof progeny virus at a concentration of 500 U/ml.

The influence of IFN-gamma on the production of progeny virus inPCV2-infected cells was determined by inoculating PK-15 cells with thestandard PCV2-stock. After the inoculation, culture medium was addedsupplemented with IFN-gamma (500 U/ml). At 0, 12, 24, 36, 48 and 72hours post inoculation (hpi) the supernatant was collected. Subsequentlythe culture was washed once with 1 ml PBS. Both the supernatant and thewashing fluid were centrifuged for 10 minutes at 15,000×g to pelletcells and debris. The centrifuged supernatant and washing fluids werecombined and considered to contain the extracellular virus. Both pelletsand cell cultures were freeze-thawed tree times and considered tocontain the intracellular virus. Intra- and extracellular virus titreswere determined by titration on PCV-negative PK-15 cells as describedpreviously.

The results of the production experiment is presented in FIG. 4. Bothextracellular and intracellular virus production was increased in theIFN-gamma treated cells compared to the non-treated control cells. Thetotal PCV2 production in cells treated with IFN-gamma at 72 hpi was 1.3log₁₀ TCID₅₀ higher compared to the non-treated cells.

Example 3 Influence of IFN-Gamma on the Expression Kinetics of PCV2Proteins

To determine the timing and localisation of PCV2 proteins expression(Capsid protein and REP), PK-15 cells were seeded on glass cover slipsand inoculated with PCV2. After inoculation, culture medium was addedwith or without 500 U/ml IFN-gamma. At 0, 12, 24, 36, 48 and 72 hpi thecells were fixed in methanol at −20° C. Afterwards, a tripleimmunofluorescence staining was performed to visualize both viralproteins and the cell nucleus. PCV2 capsid protein was detected usingpurified biotinylated porcine polyclonal anti-PCV2 immunoglobulins whichonly react with PCV2 capsid proteins. Bound porcine immunoglobulins werevisualized with streptavidin conjugated Texas Red (molecular probes,Leiden, The Netherlands). In a second step, the REP protein was detectedwith a specific mouse monoclonal antibody (F210) visualized withgoat-anti-mouse FITC (molecular probes, Leiden, The Netherlands). In afinal step, the nuclei of the cells were visualized with Hoechst 33342(Molecular Probes, Oregon, USA) at a concentration of 0.1 mg/ml. Thenumber of cells with capsid and/or REP protein was counted and thelocalisation of viral antigens was monitored by fluorescence microscopy.

Treatment of PK-15 cells did not influence the sequence of eventsconsidering the expression of PCV2 proteins. Capsid and REP proteinswere detected for the first time at respectively 12 and 24 hpi intreated and non-treated cells. The first nuclear localisation of bothproteins was observed at 24 hpi in both cultures in a similar proportionof cells.

Example 4 Influence of IFN-Gamma on the Infectious Cycle of PCV2

Since IFN-gamma induced the most constant increase in number of positivecells both in PK-15 cells as in 3D4/31 cells, this cytokine was selectedfor studies on the influence of interferons on the infectious cycle ofPCV2.

a) Influence of IFN-Gamma on the Attachment of PCV2 Capsids on 3D4/31Cells

The attachment of PCV2 capsids onto untreated 3D4/31 cells and 3D4/31cells pre-treated with IFN-gamma was studied as described by Misinzo etal. (J Gen Virol. conditionally accepted for publication). Briefly,treated and untreated 3D4/31 cells were chilled on ice and washed beforePCV2 capsids were added and allowed to attach for 0, 1, 5, 10, 15, 30and 60 minutes at 4° C. Unbound PCV2 capsids were washed-off and cellswere fixed with 3% (w/v) paraformaldehyde in phosphate-buffered salinewith calcium and magnesium (PBS+) at room temperature for 10 minutes. Inorder to stain the PCV2 capsids, cells were incubated withbiotin-conjugated anti-PCV2 swine polyclonal antibodies andstreptavidin-FITC (Molecular Probes, Leiden, The Netherlands) for 1 hourat room temperature. The slides were mounted and analysed by acquisitionof digital images of stained PCV2 capsids using a Leica TCS SP2 laserscanning spectral confocal system (Leica Microsystems GmbH, Heidelberg,Germany) linked to a Leica DM/IRB inverted microscope (LeicaMicrosystems GmbH, Wetzlar, Germany). Successive images from the apex tothe base of a single cell were taken and merged. The number of PCV2capsids attached per cell was counted for ten cells at each time pointto establish their binding kinetics into 3D4/31 cells.

No differences were observed in the number of attached virion-likeparticles per 3D4/31 cell, nor on the kinetics of virion binding to thecells as presented in FIG. 5. Both in the treated and non-treated cells,the number of attached virions cells increased quickly within 5 minutesand reached a maximum at 15 minutes after incubation. Attached virionswere observed on all cells both treated and non-treated as describedbefore.

b) Influence of IFN-Gamma on the Entry of PCV2

To study the internalization of recombinant capsids on 3D4/31, cellswere washed with RPMI-1640 at 37° C. and then incubated with capsids at37° C. for 15 minutes followed by washing of unbound capsids. Cells werethen fixed with a 3% solution of paraformaldehyde (rPBS) for 10 minutesat 30, 60 and 120 minutes since the addition of capsids into the cells,washed with PBS, and permeabilized in a 0.1% Triton X-100 solution inrPBS. Capsids were stained by incubating the calls with abiotin-conjugated anti-PCV2 swine polyclonal antibody followed bystreptavidin-FITC (Molecular Probes, Eugene, Oreg.).

A significant increase in the number of internalized virions wasobserved when 3D4/31 cells were treated with IFN-gamma.

Example 5 Influence of IFN-Gamma on the Number of Infectious ParticlesRequired for the Infection of PK-15 Cells

PCV2 infectious titres are generally determined by inoculating 10-folddilution series on PK-15 cells, followed by a staining of PCV2-positivecells. In this experiment it was investigated if the treatment of PK-15cells with IFN-gamma before, during or after the inoculation, couldincrease the sensitivity of the PCV2-titration assay. PK-15 cells weretreated with IFN-gamma (500 U/ml) as described above. Subsequently, aPCV2 stock with a known infectious titre was titrated both on treatedcells and on non-treated cells. After 72 hours of incubation afterinoculation, the cells were fixed and the total number of PCV2-positivecells was determined with the IPMA as described above. The titresobtained in IFN-gamma-treated and non-treated PK-15 cells were compared.

The number of infectious particles needed for infection of PK-15 cellswas investigated by titrating a PCV2-stock of a known infectious titrein non-treated (control) and IFN-gam ma-treated cells. In non-treatedcells the titre of the stock varied between 3.8 and 4.1 log₁₀ TCID₅₀/ml.When IFN-gamma was added to the medium after the inoculation of thestock, the titre varied between 5.3 and 5.8 log₁₀ TCID₅₀/ml. Thisindicates that between 1.2 and 2.0 log₁₀ times less infectious PCV2particles were required for infection of PK-15 cells when they weretreated with 500 U/ml IFN-gamma.

Example 6 Effect of Natural Sources of Porcine Cytokines onPCV2-Infection in PK-15 Cells

Natural Sources of Porcine Cytokines

Two natural sources of interferons were included. First, peripheralblood monocytes (PBMC's) were isolated from blood of a conventional4-week-old pig by differential centrifugation on Ficoll Paque®. Thesemonocytes were cultured in medium as described earlier (Verfaillie etal. (2002) Vet Immunol Immunopathol 81, 97-112) and stimulated with 5μg/ml concanavalin A (ConA) (Sigma). Simultaneously, a culture of PBMC'swas incubated in the same medium without ConA. After 16 hours ofincubation at 37° C. in the presence of 5% CO2, the supernatant wascollected and centrifuged to remove cells (Verfaillie et al., above).The concentration of IFNγ in these supernatants was respectively 20.5U/ml and below the detection limit as determined by a porcineIFNγ-specific ELISA (Biosource, Nijvel, Belgium).

Second, 20 times concentrated bronchiolar lavage fluid (BAL-fluid) werecollected at 1 day post inoculation from gnotobiotic pigs experimentallyinoculated with porcine respiratory coronavirus (PRCV) ormock-inoculated pigs. The BAL-fluid of the PRCV-inoculated pig hadpreviously been determined to contain 174650 U/ml IFNα, 56 U/mlIFNγ, >20480 U/ml IL6 and 273 U/ml TNFα, the BAL-fluid of themock-inoculated pig contained no detectable levels of IFNα, IFNγ andTNFα and contained 87 U/ml IL6.

Influence of Natural Sources of Porcine Cytokines on the Total Number ofPCV2-Infected Cells

Two-fold dilutions of the BAL-fluids of the PRCV and mock-inoculatedpigs were added to the medium of PCV2-inoculated PK-15 cells afterinoculation. Concentrations of the added BAL-fluids ranged from 1.25 to5%. Supernatants of ConA and mock-stimulated PBMC's were also added tothe culture medium of PK-15 cells after inoculation in concentrationsranging from 6.15 to 50%. After 36 hours of incubation, PK-15 cultureswere fixed and stained as described above and the number of infectedcells was determined.

The results of these experiments are shown in FIG. 6. When 5% of the 20×concentrated BAL fluids of a PRCV-inoculated gnotobiotic pig were addedto PK-15 cells after PCV2-inoculation, an increase in PCV2-positivecells of 745±39% was observed. Lower concentrations of this BAL-fluidshowed that the effect was dose-dependent. The BAL-fluid of themock-inoculated gnotobiotic pig did not influence the number ofPCV2-positive cells.

The supernatant of ConA-stimulated PBMC's also caused a dose-dependentincrease in PCV2-positive cells when it was added to the medium ofPCV2-inoculated PK-15 cells. When a concentration of 50% of thissupernatant was added to the cells, an increase of 215±22% was observed.Again the supernatant of non-stimulated PBMC's did not influence thenumber of PCV2-infected cells.

Example 7 Effect of Interferon on the Immune Response to the Vaccinationwith an Attenuated Circular ssDNA Virus

If interferons would increase the replication of PCV2 in vivo, it wouldprobably also increase the replication of attenuated PCV2 and by thatmechanism enhance the immune response of the pig to the virus.

Pigs are inoculated with PCV2 (field strain Imp-1121) at 19 days of age.These inoculated pigs are divided in 3 groups. One group containscontrol pigs (only PCV2). The second group of pigs receives recombinantIFN-gamma (100,000 Upper pig every three days). The third group containspigs that receive ConA (1.5 mg/kg every three days). At different timespost inoculation (10μ, 15, 21 days) samples are collected and thereplication of the virus is assessed.

An increase in the replication of PCV2 is detected in pigs injected withIFN-gamma or ConA compared to the control pigs. From this it can beconcluded that IFN has a similar effect on PCV2-replication in vivo.

Example 8 Effect of Endosomal-Lysosomal System Acidification Inhibitorson PCV2 Infection of PK-15 Cells

Endosomal-lysosomal system acidification was inhibited usinglysosomotropic weak bases (ammonium chloride and chloroquinediphosphate) and carboxylic ionophore monensin. All chemical compoundswere purchased from Sigma. In a first set of experiments, a two-folddilution of each inhibitor in PK-15 cells using Stoon-1010 was performedto determine the highest concentration of each inhibitor that exertedthe maximal effect without affecting cell viability. Finally, 25 mMammonium chloride, 125 μM chloroquine diphosphate and 6 μM monensin werechosen. PK-15 cells were cultivated for 24 hours and then inoculatedwith a dose of PCV2 for 1 hour at 37° C. The viral inoculum waswashed-off and cells were further incubated in culture medium with orwithout inhibitors of endosomal-lysosomal acidification for 24 hours.Then, the culture medium containing endosomal-lysosomal systeminhibitors was replaced with fresh culture medium without inhibitors.After the first cycle of PCV2 replication, at 36 hpi, cells were fixedwith methanol at −20° C. for 10 minutes. Cells were stored at −20° C.until they were stained using an immunoperoxidase monolayer assay (IPMA)as described by Sanchez et al. (2003, Vet Microbiol 95, 15-25).

Results:

The effect of endosomal-lysosomal system acidification inhibitors onPCV2 infection of PK-15 cells is shown in FIG. 7 (white bars). Agentsthat inhibit endosomal-lysosomal system acidification, ammoniumchloride, chloroquine diphosphate and monensin, enhance PCV2 infectioncompared to control.

Example 9 Synergistic Effect of Endosomal-Lysosomal System AcidificationInhibitors and IFN-Gamma on PCV2 Infection of PK-15 Cells

Endosomal-lysosomal system acidification was inhibited usinglysosomotropic weak bases (ammonium chloride and chloroquinediphosphate) and carboxylic ionophore monensin as described in Example 8in the following concentrations: 25 mM ammonium chloride, 125 μMchloroquine diphosphate and/or 6 μM monensin. PK-15 cells werepre-treated with or without 500 U/ml IFN-gamma for 24 hours before theywere inoculated with the same dose of PCV2 for 1 hour at 37° C. Theviral inoculum was washed-off and cells were further incubated inculture medium containing inhibitors of endosomal-lysosomalacidification for 24 hours. Then, the culture medium containingendosomal-lysosomal system inhibitors was replaced with fresh culturemedium without inhibitors. After the first cycle of PCV2 replication, at36 hours post-inoculation cells were fixed with methanol at −20° C. for10 minutes. Cells were stored at −20° C. until they were stained usingan immunoperoxidase monolayer assay (IPMA) as described above. Thenumber of infected cells per well in mock-treated cells inoculated withan equal dose of PCV2 as treated cells was used as the referent, and allresults are expressed as a percentage of this referent. All experimentswere performed three times, and each condition in a single experimentwas performed in duplicate.

Results:

Agents that inhibit endosomal-lysosomal system acidification, ammoniumchloride, chloroquine diphosphate and monensin, enhanced PCV2 infectionin IFN-gamma treated cells (FIG. 7, black bars). The effect ofendosomal-lysosomal system acidification inhibitors was synergistic tothat exerted by IFN-gamma, indicating that they increased PCV2 infectionvia different mechanisms.

Example 10 Synergistic Effect of Endosomal-Lysosomal SystemAcidification Inhibitors and IFN-Gamma on Progeny PCV2 Production

For progeny virus production assays, 2×10⁵ cells were seeded in everywell of 24-well cell culture plates. Cells were pre-treated with orwithout 500 U/ml IFN-gamma at 6 hours post seeding. At 24 hpost-seeding, cells were washed and inoculated with the prototype PCV2strain Stoon-1010 at an m.o.i. of 0.3 for 1 hour at 37° C. The viralinoculum was washed-off and cells were further incubated in 1 ml ofculture medium with or without 25 mM ammonium chloride, 125 μMchloroquine diphosphate and/or 6 μM monensin for 24 hours. Then, theculture medium with or without endosomal-lysosomal system acidificationinhibitors was replaced with fresh culture medium without inhibitors. At1, 24, 48 and 72 hours post-inoculation the supernatant was collected.Subsequently the culture was washed once with 1 ml PBS. Both thesupernatant and the washing fluid were centrifuged for 10 minutes at15,000×g to pellet cells and debris. The centrifuged supernatant andwashing fluids were combined and considered to contain the extracellularvirus. Both pellets and cell cultures were freeze-thawed three times andconsidered to contain the intracellular virus. Intra- and extracellularvirus titres were determined by titration on PCV-negative PK-15 cells asdescribed previously. Viral antigens were detected using an IPMA asdescribed above and PCV2 titre was expressed as log₁₀ TCID₅₀ per 10⁵cells.

Results:

PCV2 production was increased by treatment of the cells withendosomal-lysosomal system acidification inhibitors and/or IFN-gammatreatment. A synergistic increase in virus production was observed whencells were treated with a combination of endosomal-lysosomal systemacidification inhibitors and IFN-gamma (Table 1).

The total PCV2 virus titres in PK-15 cells treated with interferon-gammaand inhibitors of endosomal-lysosomal system acidification is providedin Table 1 and illustrated in FIG. 8.

TABLE 1 Total PCV2 virus titres in PK-15 cells treated with or without500 U/ml IFN-gamma and/or lysosomotropic agents that inhibitendosomal-lysosomal system acidification. IFN- PCV2 titres (TCID₅₀ per10⁵ cells at gamma . . . Lysosomotropic agent (U/ml) 1 hpi 24 hpi 48 hpi72 hpi Control 0 1.9 1.5 2.9 3.1 Control 500 2.0 2.1 3.4 3.9 Ammoniumchloride 0 2.1 1.9 3.0 3.8 Ammonium chloride 500 1.6 3.0 4.0 4.8Chloroquine diphosphate 0 1.4 1.8 3.3 4.3 Chloroquine diphosphate 5001.6 2.3 4.0 4.6 Monensin 0 1.9 2.3 3.9 3.6 Monensin 500 2.0 2.8 4.6 4.8

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

What is claimed is: 1-30. (canceled)
 31. A method of improving theimmune response of a subject to a vaccine comprising an attenuatedanimal circular ssDNA virus, said method comprising, administering oneor more inhibitors of endosomal-lysosomal system acidification to saidsubject, simultaneously with or shortly before or after administeringsaid vaccine to said subject.
 32. The method of claim 31, which furthercomprises administering interferon to said subject, simultaneously withor shortly before or after administering said vaccine.
 33. The methodaccording to claim 31, wherein said animal circular ssDNA virus is aCircovirus.
 34. A vaccine comprising an attenuated animal circular ssDNAvirus and one or more inhibitors of endosomal-lysosomal systemacidification.
 35. The vaccine of claim 34, which further comprises aninterferon.
 36. A vaccine comprising an attenuated animal circularssDNAvirus and interferon.
 37. An undiluted medium of an in vitro culture ofa cell-line comprising animal circular ssDNA virus characterized in thatit further comprises at least 2 U/ml interferon.
 38. An undiluted mediumof an in vitro culture of a cell-line comprising animal circular ssDNAvirus characterized in that it further comprises an agent capable ofinhibiting endosomal-lysosomal system acidification.